Abstract. Our goal is to develop new therapies for chronic GVHD (cGVHD). Our preclinical data led to 5 drug trials (1 FDA approved) in this grant period. Since most steroid-refractory cGVHD patients showed partial responses, new strategies based on deeper mechanistic insights are needed. We first showed that germinal centers (GCs) produce pathogenic Ab in cGVHD, supported by T follicular helper (Tfh) and Th17 and inhibited by T follicular regulatory (Tfr) cells. We discovered that cGVHD is restrained by donor bone marrow (BM)-derived Tfrs that have a unique phenotypic and transcriptomic signature, which may impact approaches to increase Tregs and Tfrs. Our data suggest niche cytokines/molecules supporting activated Tregs in the BM may optimize Tfrs. GC cells express PD-1. Strikingly, PD-1 blockade was effective in treating cGVHD; Tfhs were decreased and Tfrs increased, extending our findings that PD-1 inhibits Tfr generation and function. Dr. Sharpe generated mice with PD-1 immunotyrosine-based ITIM and ITSM mutations. PD-1 KO and ITSM, but not ITIM, mutant donor T cells caused high lethality, which may be due to higher T cell expansion or Tfh generation. Our cellular analyses suggest non-redundant functions in controlling Tfh and Tfr generation and function; thus, PD-1 ITSM and the ITIM signaling motifs may play distinct roles in different T cell subsets and cGVHD. Tregs and Teffector cells can express PD-1 and CD112R co-inhibitory receptors. CD112Rpos vs neg Tregs are more suppressive (Proj 2) and CD8 exhausted cells are CD112Rhi (Proj 1). CD101 is highly expressed on terminally exhausted CD8 cells (Proj 1). CD101pos vs neg Tregs are more potently suppressive and can control autoimmunity. We observed CD112R and CD101 are more highly expressed on BM vs splenic Tregs. As many Tfrs are from donor BM in our cGVHD model, BM CD112R+ or CD101+ Tregs may be key Tfr sources. We hypothesize that donor Treg site of origin controls different cGVHD phases and PD-1/CD112R or CD101 synergy is key in regulating GC facilitator and suppressor balance. Our aims will test the hypotheses that: 1. cGVHD severity is regulated by the nature of PD-1 signals in cells contributing to the GC response. 1A will test the hypotheses that inducible conditional PD-1 deletion in donor BM B cells or Tregs will decrease GC B cells and increase Tfr function, while donor T cell graft conditional PD-1 deletion in T cells or Tregs will increase pathogenic cells and cGVHD. 1B will test the hypotheses that PD-1, ITSM, or ITIM motifs have distinct functions that regulate cGVHD dependent on target cells and signaling pathways engaged early or later in cGVHD. 2. The net effects of Tfh and Tfr donor origin/function and novel inhibitory receptors (CD112R; CD101) regulate cGVHD. 2A will examine contribution of GC populations on cGVHD progression; 2B will test the hypothesis that BM Tregs are functionally distinct, denoted by CD112R or CD101, and examine their individual effects and crosstalk with PD-1 to identify mechanisms that control cGVHD that may lead to new cGVHD therapeutics.